Adenovirus-mediated gene therapy of experimental gliomas

The efficacy of adenovirus (ADV)-mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication-defective ADV carrying the herpes simplex virus thymidine kinase (HSV-tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV-tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV-tk (ADV-tk) gene or a control ADV vector containing the β-galactosidase (ADV-βgal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV-βgal and ganciclovir or with ADV-tk and saline had large tumors. No tumors were detected in animals treated with ADV-tk and with ganciclovir at doses ≥80mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV-tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV-tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV-mediated transfer of HSV-tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors. © 1994 Wiley-Liss, Inc.     

Adenoviral gene transfer to the injured spinal cord of the adult rat

We have investigated gene transfer to the injured adult rat spinal cord by the use of a recombinant adenovirus. 105 or 5 x 106 plaque-forming units (pfu) of a replication-defective adenoviral vector carrying the green fluorescent protein (GFP) reporter gene were injected into a dorsal hemisection lesion at spinal level T8. Gene expression and inflammatory responses were studied 4, 8 and 21 days after surgery. Numerous cells within 3 mm on each side of the lesion were found to express high levels of GFP at 4 days after infection as shown by GFP fluorescence and immunohistochemistry. At 8 days, expression was still strong although weaker than at 4 days. After 21 days, transgene expression had almost ceased. Expression was neither higher nor more prolonged in animals that had received the higher vector dose. Delayed injection 1 week after spinal injury also did not increase transgene expression. Infected cell types were identified immunohistochemically. The most prominent transduced cells were spinal motoneurons. Additionally, we could identify other neurons, astrocytes, oligodendrocytes and peripheral cells infiltrating the lesion site. The glial and inflammatory reaction at and around the lesion was studied by cresyl violet histology, alpha-GFAP, OX42 and alpha-CD-8 immunohistochemistry. No significant differences from controls were found in the low virus group; in the high virus group a strong invasion of CD-8-positive lymphocytes was found. Open-field locomotion analysis showed virus-infected animals performing as well as control animals. Adenoviral gene transfer may be an efficient way to introduce factors to the injured spinal cord in paradigms of research or therapy.     

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9-T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.     

Adeno-associated viral vector gene expression in the adult rat spinal cord following remote vector delivery

The current investigation tests whether adeno-associated viral vectors (rAAV) undergo remote delivery to the spinal cord via peripheral nerve injection as previously demonstrated with adenoviral vectors. The sciatic nerves of adult rats (n = 10) were injected with either an rAAV (rAAVCMV-lacZ) or adenoviral (AdCMV-lacZ) vector (1.4 x 10(7) particles/ml). After 21 days, the rAAV group demonstrated significantly higher spinal cord viral expression than the adenoviral group (P < 0.024). A second group of rats was injected with rAAV expressing the green fluorescence protein (GFP) reporter gene. GFP was detected 21 days after unilateral sciatic nerve injection in the neurons of the dorsal root ganglion and spinal cord. The codistribution of the viral genome and transgene in CNS neurons was confirmed with in situ hybridization. In summary, rAAV genes are expressed in CNS neurons following peripheral nerve injection at levels exceeding those seen following remote adenovirus injection.     

Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury

After an unilateral lesion of the corticospinal tract (CST) at the level of the medulla over-expression of Neurotrophin-3 (NT-3) in lumbar spinal cord motoneurons induced axonal sprouting of the intact CST in the acutely injured but not uninjured or chronically injured spinal cord in rats. This suggested that processes associated with immune-mediated wound healing may act with NT-3 to induce neuroplasticity. To test whether immune processes were involved we measured NT-3-induced axonal sprouting in immunosuppressed compared to immunocompetent rats. Rats were immunosuppressed with anti-leukocyte antibodies 1 day before receiving a CST lesion and then 2 weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene targeted to the motoneurons by retrograde transport. At 35 days post-lesion no axonal sprouting was measured in immunosuppressed rats whereas axonal sprouting was measured in the immunocompetent rats. We then tested whether re-evoking an immune response in chronically lesioned rats would induce neuroplasticity. Rats received CST lesions and then 4 months later were treated with systemic injections of lipopolysaccharide (LPS) 7 days before NT-3 was over-expressed in the lumbar spinal motoneurons. Axonal sprouting was observed in the LPS treated rats but not in control animals that were not treated with LPS. Further studies showed that lesioning the CST activated and LPS reactivated microglia and CD4(+) T-cells in the acutely lesioned and chronically lesioned rats, respectively. However, immunosuppression only decreased the number of activated CD4(+) T-cells suggesting they were responsible for the support of axonal growth. These observations demonstrate that processes associated with immune-mediated wound healing play a role in NT-3-induced neuroplasticity after injury.     

Effects of puromycin on incorporation of [H]lysine into protein following hemisection of rat spinal cord

Single applications of puromycin into the lesion site of spinal hemisected rats were previously shown to induce transient nerve fiber growth into the lesion. We determined the protein incorporation of [³H]lysine into rat spinal cord and brain treated with puromycin in groups of animals 3 h, 12 h, and 1, 3, 7, and 14 days after a left spinal cord hemisection at T2 and compared it to that in saline-treated controls. Each group consisted of two control animals, in which a left spinal hemisection was made at T2 and a Gelfoam sponge soaked in saline implanted in the lesion, and five experimental animals which were implanted with Gelfoam soaked in 1 mm puromycin in saline. One hour prior to utilization, animals were injected subcutaneously with 200 μCi l-[4,5(n)-³H]lysine monohydrochloride. In brain, no hemispheric or regional differences in protein or soluble fraction radioactivity could be detected in puromycin-treated or control animals. In spinal cord, inhibition of amino acid uptake was not demonstrable 3 h after hemisection, but could be detected at the site of the lesion 6 and 12 h after implantation. Three and 7 days after spinal hemisection, puromycin-treated animals exhibited a larger uptake of amino acid into protein at the lesion site than controls. The results suggest that morphological effects produced by puromycin on spinal cord regeneration may correspond to chemical events occurring within the first 24 h after cord hemisection and puromycin implantation.     

Adenovirus-mediated retrograde transfer of neurotrophin-3 gene enhances survival of anterior horn neurons of twy/twy mice with chronic mechanical compression of the spinal cord

Chronic mechanical compression of the spinal cord causes neural tissue damage, including loss of anterior horn cells around the level of injury. Exogenous delivery of neurotrophins to neuronal cells could provide neuroprotection to a spinal cord subjected to mechanical injury. We investigated the efficacy of retrograde gene delivery of adenoviral vector (AdV) carrying neurotrophin-3 (NT-3) gene into twy (twy/twy) mouse spinal cord anterior horn neurons with chronic and progressive mechanical compression at C1-C2 level. AdV-NT-3 was used for retrograde delivery via the sternomastoid muscle to the cervical spinal accessory motoneurons in 16-week-old adult twy mice with relatively mild spinal cord compression. Four weeks after the AdV-NT-3 or AdV-beta-galactosidase cDNA (LacZ) as a marker gene injection, the compressed cervical spinal cord was examined histologically, immunohistologically, and by immunoblot analysis. Immunoreactivity to NT-3 was significantly enhanced in the AdV-NT-3-injected twy mice compared with the AdV-LacZ-injected mice. The numbers of anterior horn neurons of Nissl-, choline acetyltransferase (ChAT)-, and trkC-stained and wheat germ agglutinin-horseradish peroxidase (WGA-HRP)-labeled neurons at the spinal cord level with maximum compression were significantly higher in AdV-NT-3-transfected than in AdV-LacZ-transfected twy mice. Retrograde NT-3 gene transfer to twy mouse anterior horn neurons increased neurite axonal length and arborization of WGA-HRP-labeled neurons. Our results suggest that targeted retrograde NT-3 gene delivery is feasible in the intact animal and that it enhances neuronal survival even under chronic mechanical compression of the spinal cord.     

Effects of puromycin on incorporation of [3H]lysine into protein following hemisection of rat spinal cord

Single applications of puromycin into the lesion site of spinal hemisected rats were previously shown to induce transient nerve fiber growth into the lesion. We determined the protein incorporation of [³H]lysine into rat spinal cord and brain treated with puromycin in groups of animals 3 h, 12 h, and 1, 3, 7, and 14 days after a left spinal cord hemisection at T2 and compared it to that in saline-treated controls. Each group consisted of two control animals, in which a left spinal hemisection was made at T2 and a Gelfoam sponge soaked in saline implanted in the lesion, and five experimental animals which were implanted with Gelfoam soaked in 1 mm puromycin in saline. One hour prior to utilization, animals were injected subcutaneously with 200 μCi l-[4,5(n)-³H]lysine monohydrochloride. In brain, no hemispheric or regional differences in protein or soluble fraction radioactivity could be detected in puromycin-treated or control animals. In spinal cord, inhibition of amino acid uptake was not demonstrable 3 h after hemisection, but could be detected at the site of the lesion 6 and 12 h after implantation. Three and 7 days after spinal hemisection, puromycin-treated animals exhibited a larger uptake of amino acid into protein at the lesion site than controls. The results suggest that morphological effects produced by puromycin on spinal cord regeneration may correspond to chemical events occurring within the first 24 h after cord hemisection and puromycin implantation.     

Application of recombinant adenovirus for in vivo gene delivery to spinal cord

One strategy for treating spinal cord injury is to supply damaged neurons with the appropriate neurotrophins either by direct delivery or by transfer of the corresponding genes using viral vectors. Here we report the feasibility of using recombinant adenovirus for in vivo gene transfer in spinal cord. After injection of a recombinant adenovirus carrying a β-galactosidase (β-gal) reporter gene into the mid-thoracic spinal cord of adult rats, transgene expression occurred not only in several types of cells around the injection site but also in neurons whose axons project to this region from rostral or caudal to the injection site. Among labeled neurons were those of the red nucleus, the vestibular nuclei, reticular formation, locus coeruleus, and Clarke's nucleus. A non-specific immune reaction, which could be blocked by immunosuppression with Cyclosporin A, reduced the number of transduced cells surviving at the injection site by 1 month. In neurons away from the injection site, where the immune response was minimal, transgene expression lasted for at least 2 months. These results support the idea that recombinant adenovirus can be used in the spinal cord for in vivo delivery of therapeutic genes important for supporting neuron survival and axon regeneration.     

Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo

Adult rat retinal ganglion cells undergo degeneration after optic nerve transection. Repeated intraocular injection of glial cell-line derived neurotrophic factor (GDNF) has been shown to be efficient in enhancing retinal ganglion cell survival following optic nerve axotomy. In the present study we evaluated the potential survival-promoting effect of adenovirally administered GDNF on axotomized retinal ganglion cells. A single intravitreal injection [7 x 107 plaque-forming units (pfu) or 7 x 108 pfu] of an adenoviral vector expressing the rat GDNF gene from a cytomegalovirus promoter enhanced retinal ganglion cell survival 14 days after axotomy by 67 and 125%, respectively, when compared to control animals. Intraocular administration of the vector rescued 12.6 and 23%, respectively, of the retinal ganglion cells which would otherwise have died after axotomy. An increase in retinal GDNF protein and specific virally transduced GDNF mRNA expression was detected following intraocular vector application. Our data support previous findings showing that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced retinal ganglion cell death in vivo and may constitute a relevant strategy for future treatment in traumatic brain injury and ensuing neurodegeneration.     

9L-rIL12-TK大鼠胶质瘤基因免疫疫苗的安全可控性体内实验研究

目的评估9L-rIL12-TK脑胶质瘤基因免疫活细胞疫苗在体内的安全可控性。方法将40只裸鼠随机分成3组,分别于右侧腋部皮下接种9L-rIL12-TK细胞（n=14）、9L-TK细胞（n=14）和9L细胞（n=12）,7d后将各组裸鼠分成两亚组,实验组（n=8,8,6）给予更昔洛韦（GCV）干预实验,对照组（均n=6）给予生理盐水注射,连续7d;观察裸鼠瘤体生长情况和生存时间,60d后取瘤结节及全身脏器行组织学检查（苏木精-伊红染色）。结果①给予GCV干预后7d,9L-rIL12-TK实验组及9L-TK实验组裸鼠瘤体平均体积较相应对照组及9L组显著性缩小（均P〈0.05）。60d时,8只9L-rIL12-TK实验组裸鼠瘤体均完全消退,且均无复发;8只9L-TK实验组裸鼠中,瘤体完全消退3只,瘤体缩小后再次增大5只;余亚组瘤体均呈增大趋势。②GCV干预后,9L-rIL12-TK实验组和9L-TK实验组与相应对照组比较,生存时间明显延长（P〈0.05）。③9L-rIL12-TK实验组和9L-TK实验组无瘤裸鼠接种部位无异型性肿瘤细胞,余裸鼠瘤体内均见异型性肿瘤细胞;所有裸鼠相关脏器病理学检查均未见肿瘤远处转移征象。结论9L-rIL12-TK大鼠胶质瘤细胞基因免疫疫苗在裸鼠体内可得到安全有效的控制,使制备免疫原性强且安全有效的胶质瘤基因免疫活细胞疫苗策略成为可能;其基因免疫治疗效应有待进一步研究。     

Effects of pro-inflammatory cytokines in experimental spinal cord injury

Following injury to the spinal cord, secondary tissue damage leading to massive additional tissue loss and inflammatory reactions as well as scar formation takes place. The precise functions and effects of the inflammatory cells and their secreted factors are largely unclear. The present study investigates whether the exogenous local administration of pro-inflammatory cytokines to mice after spinal cord injury can influence these intrinsic processes. A mixture of murine recombinant interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor α (TNFα) was administered to the lesioned spinal cord of adult mice. These cytokines provoked an increased recruitment and activation of macrophages and microglial cells in the lesion area when administered 1 day post lesion. In contrast, when administered 4 days after the lesion, recruitment of macrophages was slightly increased while activation of microglia was decreased as compared to controls. The amount of tissue loss 7 days after trauma was smaller in the animals receiving the cytokine mixture than in the mice receiving Ringer control solution on day 4 after lesion. Thus the role of the inflammatory response in spinal cord injury seems to be complex and well regulated. Anti-inflammatory cytokines and factors probably also contribute to the outcome of the damage following injury to the spinal cord.     

[H]Lysine incorporation into protein of hemisected, cycloheximide-treated spinal cord

A single application of a protein synthesis inhibitor, puromycin, in the lesion site of a spinal hemisection transiently decreases protein incorporation for less than 24 h and results in axonal sprouting and dendritic and synaptic stability for at least 60 days. In this study we characterized the protein-altering properties of single applications of a different protein synthesis inhibitor, cycloheximide, for comparison. The protein incorporation of [³H] lysine into hemisected rat spinal cord (left side, T2) treated with cycloheximide (100 μg/ml in Gelfoam sponge placed in wound at time of lesion) was tested 3 h, 6 h, 12 h, 1, 3, 7, and 14 days later and compared with that in 0.9% saline in Gelfoam implants as normal controls (N = 54). One hour prior to utilization, the animals were injected subcutaneously with 200 μCi l-[4,5(n)-³H]lysine monohydrochloride. No treatment-related differences in brain radioactivity were noted at any time postoperation. In spinal cord, inhibition of amino acid uptake by cycloheximide was not demonstrable at 3 h after hemisection, but could be detected from 6 h to 1 day after implantation. During that time, the TCA-soluble radioactivity at the lesion site was greater than in control animals (P < 0.05). These results suggest that as with puromycin, morphologic effects produced by cycloheximide on spinal cord regeneration may correspond to chemical events which occurred within the first 24 h after cord hemisection and cycloheximide treatment.     

Therapeutic value of 21-aminosteroid U74389F in acute spinal cord injury

Abstract

The effect of bolus injections of 21-aminosteroid U74389F after an acute spinal cord compression trauma in rats was studied. Cortical somatosensory evoked potentials (CSEPs) were recorded before and after a weight-induced injury of 120 g and monitored up to five hours post-injury. All U74389F treatments were given as i.v. bolus injections of 15, 7.5, and 3.75 mg kg^–1 at 1, 2, 3 h after the trauma, respectively. The CSEPs were abolished immediately after the injury in the control and treated animals. The majority of the treated animals (88.8%) demonstrated a return of the CSEPs within the second hour post-injury. In contrast, the animals in the control group showed only 44.4% recovery at this time period. At three hours post-injury, U74389F-treated animals (n = 18) showed a full recovery (100%) while the recovery rate remained at 44.4% for the control animals. We conclude that the bolus administration of U74389F one hour after injury facilitates the return of the spinal cord function as measured by the CSEPs in this compression model of acute spinal cord trauma. [Neurol Res 1993; 15: 321-326]     

Cellular monstrosity in embryonic neuronal neoplasm produced by human adenovirus in rats

Summary Solid medullary brain and spinal cord neoplasms developed in all 10 offspring of an outbred Sprague-Dawley rat, between 37 and 99 days after a single postnatal (within 24 hrs) inoculation of 0.05 ml of human adenovirus type 12, 10^3.5–10^4.5 TCID₅₀ HeLa cells/0.1 ml in the left frontal lobe. Seven rats developed multicentric neoplasms in both hemispheres and in peri-aqueductal areas of the brain stem, one of which was associated with an incipient spinal cord tumor in the sacral segment. One rat developed a solid tumor involving the right parieto-occipital region. The remaining two cases were solid spinal cord tumors arising from the dorsal half of the thoracolumbar segments. The remarkably uniform microscopic appearance was designated as a counterpart of human embryonic neuronal neoplasms. Characteristic neuronal and multinucleated giant cells emerged throughout the tumor tissue with argentaffine, neurofibril-like cytoplasmic expansions and a unique cilium (a 9+0 pattern of tubules) associated with a pair of centrioles. This cilium morphology was also a hallmark of the majority of tumor cells that formed characteristic pseudorosettes. The occasional emergence of two sets of cilia and centrioles in monstrous cells suggested probable modes of cytogenesis in relation to cessation of abnormal cell division.     

Endogenous gamma interferon produced in central nervous system by systemic infection infection with Theiler's virus in mice

Theiler's virus GD VII strain causes acute encephalomyelitis by intracerebral inoculation. We established acute encephalomyelitis in mice by the intravenous (i.v.) inoculation of Theiler's virus GD VII strain. Replication of Theiler's virus injected i.v. could be observed in both the brain and spinal cord of mice, and interferon (IFN)-γ could be detected in the extracts of brain and spinal cord in parallel with viral replication. Furthermore, by the injection of anti-IFN-γ monoclonal antibody (mAb) on Day 1 post-infection (p.i.), mortality and virus titres in the spinal cord increased compared with the control mice treated with normal rat globulin. The histological exacerbation of inflammation was observed in spinal cord of anti-IFN-γ mAb-treated mice. These results indicate that endogenous IFN-γ, produced locally in the brain and spinal cord of mice through both antiviral action and anti-inflammatory action of IFN-γ in central nervous system, plays an important role in Theiler's virus infection.     

腺病毒介导HSV-tk和反义IGF-1联合基因治疗鼠脑胶质瘤

目的　研究腺病毒介导 Ｈ Ｓ Ｖｔｋ／ Ｇ Ｃ Ｖ 系统和反义 Ｉ Ｇ Ｆ１ 联合基因治疗大鼠脑胶质瘤,并对其机制作初步分析。方法　利用腺病毒为载体观察联合 Ｈ Ｓ Ｖｔｋ／ Ｇ Ｃ Ｖ 和反义 Ｉ Ｇ Ｆ１ 离体杀伤 Ｃ６细胞的效果, 并在大鼠脑内接种 Ｃ６ 细胞第８ 天进行 Ｈ Ｓ Ｖｔｋ／ Ｇ Ｃ Ｖ 和反义 Ｉ Ｇ Ｆ１ 的原位治疗, 观察大鼠生存期变化。结果　 Ｇ Ｃ Ｖ 对转染反义 Ｉ Ｇ Ｆ１ 基因和ｔｋ 基因的 Ｃ６ 细胞较转染ｔｋ 基因的 Ｃ６ 细胞敏感。联合应用 Ｈ Ｓ Ｖｔｋ／ Ｇ Ｃ Ｖ 系统和反义 Ｉ Ｇ Ｆ１ 原位治疗脑肿瘤比单用任何一种治疗方法效果明显, 免疫组化发现联合基因治疗组肿瘤有大量 Ｃ Ｄ＋４ 、 Ｃ Ｄ＋８ 淋巴细胞浸润。结论　反义 Ｉ Ｇ Ｆ１ 基因可增强 Ｈ Ｓ Ｖｔｋ／ Ｇ Ｃ Ｖ 杀伤肿瘤的作用。     

Neurotrophic factors expressed in both cortex and spinal cord induce axonal plasticity after spinal cord injury

We reported recently that overexpression of neurotrophin-3 (NT-3) by motoneurons in the spinal cord of rats will induce sprouting of corticospinal tract (CST) axons (Zhou et al. [2003] J. Neurosci. 23:1424-1431). We now report that overexpression of brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF) in the rat sensorimotor cortex near the CST neuronal cell bodies together with overexpression of NT-3 in the lumbar spinal cord significantly increases axonal sprouting compared to that induced by NT-3 alone. Two weeks after unilaterally lesioning the CST at the level of the pyramids, we injected rats with saline or adenoviral vectors (Adv) carrying genes coding for BDNF (Adv.BDNF), GDNF (Adv.GDNF) or enhanced green fluorescent protein (Adv.EGFP) at six sites in the sensorimotor cortex, while delivering Adv.NT3 to motoneurons in each of these four groups on the lesioned side of the spinal cord by retrograde transport from the sciatic nerve. Four days later, biotinylated dextran amine (BDA) was injected into the sensorimotor cortex on the unlesioned side to mark CST axons in the spinal cord. Morphometric analysis of axonal sprouting 3 weeks after BDA injection showed that the number of CST axons crossing the midline in rats treated with Adv.BDNF or Adv.GDNF were 46% and 52% greater, respectively, than in rats treated with Adv.EGFP or PBS (P < 0.05). These data demonstrate that sustained local expression of neurotrophic factors in the sensorimotor cortex and spinal cord will promote increased axonal sprouting after spinal cord injury, providing a basis for continued development of neurotrophic factor therapy for central nervous system damage.     

Regeneration of corticospinal axons in the rat.

In the rat, a few long descending motor tracts capable of carrying an impulse and causing a propagated impulse in the ipsilateral sciatic nerve will regenerate after complete spinal cord transection. In this experiment such regeneration was found in both treated and control animals. Orthograde axonal transport of tritiated proline injected into the motor cortex labels only the corticospinal tracts in the rat spinal cord. Scintillation counts of measured lengths of spinal cord can be used as a measure of the number of labeled corticospinal axons. Comparison of radioactivity per unit length of measured cord segments taken from above and below the site of a previous spinal cord transection can give a reliable estimate of the number of labeled axons that regenerated and crossed the site of injury. Using this test we have demonstrated that some corticospinal axons had regenerated six months after spinlal cord transection in control animals, animals made tolerant to degenerating spinal cord antigens, and animals treated with cyclophosphamide. A group treated with a single 75 mg per kilogram dose of cyclophosphamide 24 hours after spinal cord transection showed the best evidence of corticospinal tract regeneration.     

Physiological, biochemical and histological changes in skeletal muscle, neuromuscular junction and spinal cord of rats rendered paraplegic by subarachnoidal administration of 6-aminonicotinamide

Exposure of rats to the nicotinamide analog, 6-aminonicotinamide, produces an irreversible rigid paralysis of the hind limbs. The present study describes electrophysiological alterations that occur in extensor digitorum longus (extensor) and soleus muscles of rats treated with a single subarachnoid injection of 6-aminonicotinamide and compares these changes with cellular and biochemical changes that occur in the spinal cord. Injection of 50 μ of 6-aminonicotinamide into the subarachnoid space at L2–L4 of adult female rats produced rigid paralysis of both hind limbs in 24–36 h. In extensor muscles, the membrane potential decreased significantly about 48 h after injection, but this partial depolarization took somewhat longer in the soleus muscles. Spontaneous transmitter release, as studied by frequency and amplitude of miniature endplate potentials, was significantly affected from day 7 after injection. At the onset of paralysis, 6-phosphogluconate levels were increased more than 1000-fold in the lumbar region of the spinal cord and returned to control levels by 14 days after injection. This metabolite thus serves as a biochemical marker for the presence of the 6-aminonicotinamide analog of NADP in tissues. Beginning at 3 days an increase in small phagocytic cells was noted in both the cervical and lumbar regions of the cord. These proliferating cells were highly localized to the anterior horn region where maximal numbers were detected at 8 days. Few viable motor neurons were present in the lumbar region of the cord at this time. There was a close correlation between the accumulation of small cells and the increased β-glucuronidase activity. Despite the marked cellular alterations in the spinal cord at 8 days there were no changes in the total extractable lipid in gray and white matter. Cellular changes were maintained in the lumbar region for as long as 540 days. At this time most of the electrophysiological properties of the extensor muscle were restored to normal, suggesting that collateral sprouts from the residual motor neurons may have reinnervated the denervated muscle fibers.